Splice Variants of the Dual Specificity Tyrosine Phosphorylation-regulated Kinase 4 (DYRK4) Differ in Their Subcellular Localization and Catalytic Activity
ABSTRACT Dual specificity tyrosine phosphorylation-regulated kinases, DYRKs, are a family of conserved protein kinases that play key roles in the regulation of cell differentiation, proliferation, and survival. Of the five mammalian DYRKs, DYRK4 is the least studied family member. Here, we show that several splice variants of DYRK4 are expressed in tissue-specific patterns and that these variants have distinct functional capacities. One of these variants contains a nuclear localization signal in its extended N terminus that mediates its interaction with importin α3 and α5 and that is capable of targeting a heterologous protein to the nucleus. Consequently, the nucleocytoplasmic mobility of this variant differs from that of a shorter isoform in live cell imaging experiments. Other splicing events affect the catalytic domain, including a three-amino acid deletion within subdomain XI that markedly reduces the enzymatic activity of DYRK4. We also show that autophosphorylation of a tyrosine residue within the activation loop is necessary for full DYRK4 kinase activity, a defining feature of the DYRK family. Finally, by comparing the phosphorylation of an array of 720 peptides, we show that DYRK1A, DYRK2, and DYRK4 differ in their target recognition sequence and that preference for an arginine residue at position P -3 is a feature of DYRK1A but not of DYRK2 and DYRK4. Therefore, we highlight the use of subcellular localization as an important regulatory mechanism for DYRK proteins, and we propose that substrate specificity could be a source of functional diversity among DYRKs.
- SourceAvailable from: Jean M Delabar
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- "Kinase activity of DYRK1A protein was determined from hippocampus (6 mice per group) according to previously published protocol  "
ABSTRACT: Trisomy for human chromosome 21 results in Down syndrome (DS), which is among the most complex genetic perturbations leading to intellectual disability. Accumulating data suggest that overexpression of the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A), is a critical pathogenic mechanisms in the intellectual deficit. Here we show that the green tea flavonol epigallocatechin-gallate (EGCG), a DYRK1A inhibitor, rescues the cognitive deficits of both segmental trisomy 16 (Ts65Dn) and transgenic mice overexpressing Dyrk1A in a trisomic or disomic genetic background, respectively. It also significantly reverses cognitive deficits in a pilot study in DS individuals with effects on memory recognition, working memory and quality of life. We used the mouse models to ensure that EGCG was able to reduce DYRK1A kinase activity in the hippocampus and found that it also induced significant changes in plasma homocysteine levels, which were correlated with Dyrk1A expression levels. Thus, we could use plasma homocysteine levels as an efficacy biomarker in our human study. We conclude that EGCG is a promising therapeutic tool for cognitive enhancement in DS, and its efficacy may depend of Dyrk1A inhibition.Molecular Nutrition & Food Research 02/2014; 58(2). DOI:10.1002/mnfr.201300325 · 4.91 Impact Factor
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ABSTRACT: Enzymes are key molecules in signal-transduction pathways. However, only a small fraction of more than 500 human kinases, 300 human proteases and 200 human phosphatases is characterised so far. Peptide microarray based technologies for extremely efficient profiling of enzyme substrate specificity emerged in the last years. This technology reduces set-up time for HTS assays and allows the identification of downstream targets. Moreover, peptide microarrays enable optimisation of enzyme substrates. Focus of this review is on assay principles for measuring activities of kinases, phosphatases or proteases and on substrate identification/optimisation for kinases. Additionally, several examples for reliable identification of substrates for lysine methyl-transferases, histone deacetylases and SUMO-transferases are given. Finally, use of high-density peptide microarrays for the simultaneous profiling of kinase activities in complex biological samples like cell lysates or lysates of complete organisms is described. All published examples of peptide arrays used for enzyme profiling are summarised comprehensively.Molecular Biotechnology 05/2011; 49(3):283-305. DOI:10.1007/s12033-011-9402-x · 2.28 Impact Factor
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ABSTRACT: Protein kinases are fascinating biological catalysts with a rapidly expanding knowledge base, a growing appreciation in cell regulatory control, and an ascendant role in successful therapeutic intervention. To better understand protein kinases, the molecular underpinnings of phosphoryl group transfer, protein phosphorylation, and inhibitor interactions are examined. This analysis begins with a survey of phosphate group and phosphoprotein properties which provide context to the evolutionary selection of phosphorylation as a central mechanism for biological regulation of most cellular processes. Next, the kinetic and catalytic mechanisms of protein kinases are examined with respect to model aqueous systems to define the elements of catalysis. A brief structural biology overview further delves into the molecular basis of catalysis and regulation of catalytic activity. Concomitant with a prominent role in normal physiology, protein kinases have important roles in the disease state. To facilitate effective kinase drug discovery, classic and emerging approaches for characterizing kinase inhibitors are evaluated including biochemical assay design, inhibitor mechanism of action analysis, and proper kinetic treatment of irreversible inhibitors. As the resulting protein kinase inhibitors can modulate intended and unintended targets, profiling methods are discussed which can illuminate a more complete range of an inhibitor's biological activities to enable more meaningful cellular studies and more effective clinical studies. Taken as a whole, a wealth of protein kinase biochemistry knowledge is available, yet it is clear that a substantial extent of our understanding in this field remains to be discovered which should yield many new opportunities for therapeutic intervention.Bioorganic Chemistry 08/2011; 39(5-6):192-210. DOI:10.1016/j.bioorg.2011.07.004 · 2.14 Impact Factor